Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of putative inciting antigens, including viruses, chemicals, medical drugs, and tumor antigens. Whatever it?s specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its association with clonal hematologic diseases, especially paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndromes (MDS). In the clinic, studies have been directed towards more effective immunosuppression in aplastic anemia and the application of immunosuppressive regimens to related bone marrow failure syndromes. We have completed analysis of rabbit ATG, a more potent formulation antilymphocyte globulin recently licensed for use by the FDA in the US: about 1/3 of patients who have failed a course of horse ATG will respond hematologically to transfusion-independence on treatment with rabbit ATG; the majority of patients who relapse after horse ATG will respond to rabbit ATG. In patients with refractory aplastic anemia, unresponsive to horse ATG, we have also observed hematologic recovery using the monoclonal antibody CAMPATH, directed against CD52, a ubiquitous antigen on human lymphocytes. These clinical results ave led to the institution of a triple arm, randomized protocol for severe aplastic anemia on presentation; approximately 30 patients have been entered into this protocol, which compares horse ATG with a long course of cyclosporin; rabbit ATG with standard cyclosporin; and CAMPATH alone. Hematologic responses have been observed in all arms, including for CAMPATH , but more patients will be required for adequate comparison of the primary endpoints of hematologic response as well as relapse and clonal evolution. In studies in other diseases, we have established that the monoclonal antibody declizumab is effective not only in moderate aplastic anemia but also in pure red cell aplasia, leading to transfusion-independence in single lineage marrow failure in this format about 1/3 of patients, with no or minimal drug toxicity. In myelodysplasia, analysis of outcomes in a large number of patients who were treated with ATG with or without cyclosporin have shown a clear survival advantage, in comparison to historical data obtained in multi-center observations. Also in myelodysplasia, CAMPATH has been effective in several patients in a new protocol as a single agent, again with modest toxicity in comparison to ATG. The Hematology Branch has also participated in a large multi-center trial of Ecluzimab, a monoclonal antibody to the C5A component of complement; the results of this study will be published shortly. In the laboratory, efforts have concentrated on the mechanism of immune suppression, and the pathophysiology of late clonal events, including PNH, MDS, and acute leukemia. Studies of T-cell clonotypes continue to show a varied pattern in patients who respond, relapse, or are refractory to immunosuppressive therapy, although in general dominant clonal types recede with clinical improvement and recur, often accompanied by new clones of T-cells, on relapse. Current studies analyze the pattern of transcription of cytokines and immunomodulatory and other regulatory genes in isolated clones obtained by flow cytometric sorting based on the specific V-beta subfamily of the T-cell receptor. Other investigations have examined on the signal transduction pathways involved in T-cell polarization and the aberrant T-cell response. Results have including the finding of increased T-bet, a transcriptional regulator that is crucial for T-cell polarization to TH1 cells. New results, all consistent with an immune hypothesis for the pathogenesis of acquired aplastic anemia, include decreased PRF1 gene expression, low perforin, and low cytolytic activity in aplastic anemia, similar to that observed in familial hemophagocytic syndrome. Analogously, we have observed a high prevalence of decreased expression of SAP, a small immunomodulatory protein also important in the regulation of gamma interferon gene expression. In other studies relevant to the genetics of acquired aplastic anemia, we have described mutations in genes of the telomere repair complex, including for telomerase, TERT, and TERC in patients with apparently acquired aplastic anemia. In a large Mennonite kindred with a novel TERT mutation, the mutation has tracked with short telomeres and a spectrum of hematologic manifestations, including frank aplastic anemia, mild anemia, macrocytosis, and no hematologic abnormalities; the presence of the mutation can be inferred in six generations of this highly informative family; one patient with TERT mutation in this kindred suffered early liver failure, requiring liver transplantation. Studies of telomere repair in aplastic anemia have also led to the discovery that a likely mechanism of action of male hormones, historically utilized for the treatment of a variety of bone marrow failure conditions, is telomerase gene up-regulation. In primary lymphocytes and CD34 cells, different androgen preparations act through conversion by aromatase to estrodiol and binding to the estrogen receptor, leading to activation of TERT. Sex hormones can also up-regulate telomerase activity in cells from patients mutant in TERT. We are investigating the functional role in the Shwachman-Bodian-Diamond syndrome gene (SBDS), which we found to be mutated in heterozygosity in patients with apparently acquired aplastic anemia and in their mothers with life-long mild macrocytic anemia. The SBDS gene product does not associated physically with the telomere repair complex; heterogeneity of telomere length in DNA hybridization and other data suggests that SBDS may be related to an alternative pathway of telomere repair. In the area of clonal evolution, we have focused on two cytogenetically defined forms of myelodysplasia, trisomy 8 and monosomy 7 MDS. In trisomy 8 MDS, patients are frequently responsive to immunosuppressive therapy and their blood counts may be dependent on low level continued cyclosporin. In previous work, we demonstrated that trisomy MDS is similar to aplastic anemia in a high prevalence of oligoclonal T-cells, which appear to be specifically reactive to the cytogenetically aberrant clone. In more recent work, we have demonstrated a block in the apoptosis pathway: trisomy 8 cells, while annexin-positive, do not undergo DNA degradation and are capable of proliferation in vitro. This block has correlated with increased gene expression of c-myc, cyclin D1, and surviving. Most recently, the antigen in trisomy 8 has been shown to Wilms tumor-1 (WT-1), previously implicated in acute myelogenous leukemia and the subject of vaccine development in studies of malignant hematology. T-cells in a large proportion of patients with trisomy 8 are specific for WT1, as demonstrated as in tetramer methodology. In monosomy 7, the most frequent cytogenetic abnormality to evolve form aplastic anemia, clinical data have suggested an association with either the exogenous administration of granulocyte-colony stimulating factor (G-CSF) or persistent neutropenia and elevated endogenous G-CSF levels. We have shown that monosomy 7 does not arise de novo from normal bone marrow cells, but under conditions of high G-CSF stimulation, small clones of pre-existing monosomy 7 cells expand, as detected by fluorescent insitu hybridization (FISH), due to increased expression of a normal isoform of the G-CSF receptor which lacks the cytocellic signaling domain for differentiation. As a result, under conditions of high G-CSF in vivo, these cells likely are selected to undergo proliferation and not differentiation.